EP0551320A1 - Protein produced from hirudo medicinalis - Google PatentsProtein produced from hirudo medicinalis
- Publication number
- EP0551320A1 EP0551320A1 EP19910916983 EP91916983A EP0551320A1 EP 0551320 A1 EP0551320 A1 EP 0551320A1 EP 19910916983 EP19910916983 EP 19910916983 EP 91916983 A EP91916983 A EP 91916983A EP 0551320 A1 EP0551320 A1 EP 0551320A1
- Grant status
- Patent type
- Prior art keywords
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Protein from Hirudo medicinalis
The present invention relates to a novel protein from the leech Hirudo medicinalis, its muteins and their Her¬ position and use.
a number of medically interesting factors have been described that play a role in areas such as blood coagulation and fibrinolysis from the secretions of various flukes. These factors include fibrinolytic agents which degrade fibrin or fibrinogen directly and Plas activators inogen-. To the fibrin or fibrinogen-depleting substances include the hementin (of He enteria ghilianii) and the de stabilase (Hirudo medicinalis from; IP Baskova et al Folia Huh atol, Leipzig, 115, 1988, p.166..). A plasminogen activator described in secretions of Hementeria depressa.
Next also inhibitors of clotting, such are the thrombin bininhibitor hirudin (Hirudo medicinalis from), a factor Xa-in hibitor (Hirudo medicinalis out), trypsin and plasmin and chymotrypsin and elastase inhibitors such as the Bdellin or eglins described (Hirudo medicinalis out) (Leech Biology and Behavior, Vol. II, Oxford, Science Publication / RT Sawyer, 1986).
It has now been isolated from Hirudo medicinalis a new factor.
The invention relates to a novel protein from Hirudo medi¬ cinalis that prolongs the clotting time of the blood, is relatively heat stable and has a molecular weight of 25 to 34 kDa be¬ sits, and its muteins.
The novel protein prolongs the intrinsiche clotting time (APTT) and extrinsic coagulation time (PT) of the blood significantly. It differs in its coagulation-retardant properties are of hirudin and the factor Xa inhibitor. In fractions in which the APTT prolonging activity is present, neither appropriate amounts of hirudin (by ELISA) or factor
Xa inhibitor activity be detected (by color substrate). As muteins of the new protein, such proteins are understood to be derived from the new protein by substitution, deletion and / or addition of amino acids or peptides without their properties differ greatly lower the new protein.
The new protein can be from leeches (Hirudo medicinalis) win. To this end, the leeches are placed in water. After stimulation with arginine leeches enter the factor into the watery solution from which the new protein chromatography or in a known manner by ion exchange, affinity, gel permeation chelating and is isolated by hydrophobic chromatography.
You can dot the leeches after arginine stimulation and isolate the novel protein as described above from the puncture.
It is also possible to purify the new protein from the homogenate of heads dekapitierter flukes in an analogous manner.
To the protein for therapeutic purposes in large quantities to make ver¬ fügbar, can be known genetic engineering techniques (see Maniatis, T. et al, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, NY, 1989) apply. For this purpose, the protein is first produced in pure form. Subsequently, the protein is digested and the resulting peptides fractionated by reversed phase HPLC (RHPLC). Some of these peptides, the amino acid sequence is determined. With oligonucleotides derived from the determined sequences, the gene coding for the novel protein is then identified in a c-DNA library by sequence-specific hybridization filter. The genetic information thus obtained can then be put in various host cells by known methods for expression. To the expression rate pressionssystemen one hand prokaryotes such as E. coli or Bacillus subtilis, but also eukaryotes such as yeast and mammalian cells include (for example CL27 mouse fibroblasts, hamster, Insekten¬ cells).
The new protein and its muteins are useful for treating diseases, particularly for treatment and prophylaxis of thrombotic conditions. Be i sp i el
a) extraction of Egelextrakt
Extracts of Hirudo medicinalis were obtained go to the following Ver¬:
1. flukes were treated 75 min with a 10 mM arginine solution. During this time, the solution adopted by the secretory activity of the leech on a yellowish color.
After exchange of the liquid against water leeches further secreted.
2. leeches who had been first treated in accordance with arginine were punctured and won the stomach contents.
b) Purification of the new protein.
The liquids obtained in a) were individually or together for 10 minutes at 90 ° C and then cooled immediately on ice. After centrifugation (20 min, 10,000 rpm, Sorvall® SA 600 rotor) the supernatant was initially 1: 2 to 1: 5 diluted with PBS. The pH was adjusted to 7.4. The solution was then equilibrated in a PBS
Q-Sepharose® column (10 ml, Pharmacia, Sweden). After washing away unbound material, the column with a linear gradient of 0 to 100% buffer A (PBS + 1 M NaCl) was eluted. Aliquots of the fractions were assayed for APTT prolongation. The new factor eluted under these conditions is between 0.3 M and 0.5 M NaCl.
Fractions containing activity were combined, 1: 2 with 20 mM phosphate buffer, 250 mM NaCl pH 7.0 is diluted and loaded onto a Cu Chelating column (1 ml). After rinsing with 10 to 15 column volumes of equilibration buffer (20 mM NaPhosphate, 250 mM NaCl pH 7.0), elution was carried out by applying a linear gradient of 0 to 100% buffer B (20 mM succinic acid, 1 M NaCl pH 4.0) , The column was then nacheluiert with either 20 mM phosphate, 2 M NaCl, 10 mM EDTA, pH 7 or 20 mM succinic acid 2 M NaCl, 100 M histidine pH. 4 Aliquots of individual fractions fractions were assayed for APTT prolongation containing activity pooled and Centricon 10 (A icon, Cat. No. 4206), concentrated, buffer exchanged to PBS and on a FPLC Gelfilrationssäule (Superose® 12, Pharmacia) at a flow rate 0.5 ml / min purified further.
The fractions containing activity were pooled and applied to a column equilibrated with PBS mono Sepharose® (Pharmacia, Sweden). From the Mono Q column, the elution was carried out either with a pH gradient (0 to 100% B, Buffer B = 20 M succinic acid 1 M NaCl pH 4) or with a salt gradient (0 to 100% buffer A).
For further purification of the new factor chromatography on S-Sepharose (LKB, Pharmacia) was carried out. For this purpose the value of fractions of the Q-Sepharose or Cu-chelating chromatography initially using a concentrate chamber (Amicon, YM 10 membrane) at pH 4 or 7 and onzentriert by repeatedly filling and concentrating desalted.
After the final step, the concentrates were diluted to 20 mM succinic acid pH 2.5 (final concentration) and applied to a column equilibrated with 20 mM succinic acid pH 2.5 S-Sepha- rose-Säule®. possibly existing hirudin was separated by elution with 30 mM of succinic acid pH 4.5. The elution of the new factor? was performed by applying a buffer gradient by PE pH 7.5. In the value of the S-Sepharose fractions to a pH of 5.5 was - 7 measured. The fractions containing activity (thrombin inhibition) were further containing a hirudin Elisa (s. Example 6a) were tested. Fractions which prolong the APTT and were negative in the hirudin Elisa were combined, aufkonzen¬ trated, evaporated to dryness, taken up in ammonium acetate pH 6 by RHPLC on a C-4 phase (rp 304® Bio-Rad) purified further. Reversed phase HPLC (RHPLC)
The thrombin inhibition fractions comprising the S-Sepharose chromatography were further purified by reversed phase HPLC on a Bio-RAD rp304® column with a 20 M ammonium acetate, pH 6, 20 M ammonium acetate, 50% Acetonitri 1 system , For this, the fractions were concentrated to a volume of 200 to 500 .mu.l and eluted by applying a gradient of acetonitrile from RHPLC. The new factor elusiert at a Acetonitri Ikonzentration of 7.5% + _ 3%. The activity of the new Thrombininbibitors is retained and can be detected by Tricine-SDS-polyacrylamide gel electrophoresis or APTT determination.
c) Characterization of the new protein
1. Temperature stability
An aliquot of the new factor was heated for 10 min at 95 ° C and then immediately cooled on ice. The activity was determined by measuring the inhibition of the APTT. By the heat treatment, the activity decreased by about 10%.
2. solvent stability
An aliquot of the sample containing activity was added at room temperature with 50% acetonitrile in 20 M ammonium acetate pH 6.0 and incubated for 30 min. After removing the solvent, the influence of the sample was analyzed for APTT. It turned out that the new factor is not influenced in its activity by acetonitrile.
3. Protein Nature
An aliquot of the new factor was incubated with 10 ug trypsin for 16 h at 37 ° C. The reaction was stopped by boiling for 5 minutes and immediately cooling with ice and thereafter determines the influence of the sample to the inhibition of the APTT. It has also been moved to a control sample that was not otherwise treated equally with trypsin. The Ergeb¬ Results show that after incubation with trypsin, the activity of factor disappears, proving its protein nature. 4. Molecular Weight
a) The molecular weight of the new protein was limited defined using membranes pore size. For this purpose, fractions containing the new protein were incubated with buffer (PBS pH 7.2 or 20 mM succinic acid pH 4), washed in a Centricon® microconcentrator (10 and 30 kDa Ausschluß¬ limit) and 60 min at 5000 xg centrifuged. The runs of the membranes were in a Speed Vac Concen- trator to dryness, resuspended in PBS and likewise as are the residues of the membranes in the APTT measured.
At both pH values, the new protein made by Centricon 10, focused but not by Centricon 30th This means that the novel protein should have a molecular weight of 10-30 kDa.
b) The molecular weight was determined next (on a calibrated gel filtration column Superose 12). while PBS was used as eluent at a flow rate of 0.3 ml / min. There were
fractionated fractions of 150 ul and assayed for APTT prolongation. The maximum activity was determined under these conditions Bedin¬ to 34 kDa in a range of 25 kDa.
c) Determination of molecular weight by Tricine-SDS-polyacrylamide gel electrophoresis
The gel electrophoresis was performed according to the procedure (Schagger, H. and von Jagow, G .; Analytical Biochemistry, 166, 368-379 (1987) at 20 A and 1400 V, 30 W, with a power company LKB, and an overflow chamber of the " mighty s performed all system ". After completion of the electrophoresis, the gel was incubated X100 and then to a thrombin and fibrinogen-containing agarose plate 2 firstly for 30 minutes in a 2.5% solution of Triton times
issued 20 minutes. The concentration of thrombin in the agarose was adjusted such that polymerization of the fibrinogen took place within 2 to 4 hours under the action of thrombin. The presence of the new thrombin inhibitor was by unpolymerized places in the
Agarose displayed. (By staining the molecular weight marker calibration proteins: Intact Myoglobin 17.2 kDa, myoglobin I + Bromcyanpeptid Uli 14.6 kDa, myoglobin I Bromcyanpeptid 8.2 kDa, myoglobin II Bromcyanpeptid 6.4 kDa, myoglobin III Bromcyanpeptid 2.6 kDa, myoglobin 1 -14), the Molekular¬ weight of the new thrombin inhibitor with 14 6 + _ 3 kDa determined.
5. point of application of the new protein in the coagulation cascade
The new factor was overall in various assay systems tested to determine how and which factors in the coagulation as (he ade inhibited.
a) APTT (activated partial thromboplastin time ***) for Nach¬ facing an anticoagulant activity in the intrinsic coagulation cascade see.
human plasma (eg Preciclot®, Boehringer / Ma, no. 654370) was diluted samples to be tested in 50 ul. For this purpose, a 50 ul PTT reagent (Boehringer / Ma, no. 886 050) was pipetted and incubated for 5 min at 37 ° C. By adding 50 ul 25 mM CaCl the coagulation cascade has started. the time was measured until the occurrence of clots Plasma¬.
The samples showed a significant prolongation of plasma clotting time (intrinsic).
b) PT (= prothrombin time, Quick-Test) for detecting an anticoagulant effect on the extrinsic coagulation cascade.
Samples were resuspended in 100 ul human plasma (eg Preciclot®, Boehringer / Ma, no. 654370) diluted. To this was added 100 .mu.l of thromboplastin (1: 1 with NaCl 0, 9% diluted) were pipetted. With 50 ul of 25 mM CaCl 2, the coagulation cascade has started. the time was measured until the occurrence of clots Plasma¬. The samples showed a significant Ver¬ prolongation of plasma clotting time (extrinsic). c) FXa inhibition assay for differentiation of FXa inhibitor
Samples (diluted in PBS) were incubated with factor Xa (FXa) and a specific chromogenic substrate for FXa 60 min at room temperature:
25 ul of sample
50 ul FXa (Boehringer / Ma, no. 602,388, 0,025 U / ml) 100 ul S-2222 chromogenic substrate (Kabi Vitrum-, 16.9 ml H 2 0 / vial) After 5 minutes, the optical density at 405 nm first measured. After 60 min the reaction was stopped with 50 .mu.l of glacial acetic acid and the optical density at 405 nm is measured again.
As controls approaches without FXa and samples are used without inhibitor. For the evaluation, a Δ OD 405 was 40 'is calculated using the following formula:
Δ OD = CθD 60m-OD5 min] + FXa - [OD OD 5minJ' 0min- 6-FXa
(Difference in absorbance with and without FXa).
From respective batches with and without inhibitor, the degree of inhibition of FXa is given by (in%)
Δ 0D + inhibitor
% Inhibition = 100 x
Δ 0D -lnhibitor
d) thrombin time
A sample of the new protein was diluted in 100 .mu.l ver¬ plasma. By adding 50 ul thrombin reagent (Boehringer / Ma, no. 126 594) was started clotting. The time to onset of plasma clots was determined turbine-do etrisch.
The evaluation was performed using a calibration curve with known thrombin inhibitors (eg, hirudin). The results of obtained show that the new protein is a thrombin inhibitor. 6. Attempts to define the new protein to hirudin
a) fractions of the new protein, the ATU showed an APTT prolongation according 2500, were introduced into a Hirudin ELISA:
Microtiter plates (Flow 77-173-05) - streptavidin-peroxidase Ko plex (Boehringer / Ma 1,089,153) tetramethylbenzidine (Serva 35926) Peroxidase substrate: 0.1 ml TBM solution (42 mM in DMSO TBM) and 10 ml of substrate buffer (0, M Na-acetate pH 4.9) to mix slowly 1; then addition of 14.7 .mu.l of 3% H2O2
Coat microtiter plates with 0.2 ml / well hirudin
(1 ug / ml, in 0.1 M NaHC0 3, pH 8.3) - saturate with 1% BSA / PBS; 0.3 ml / well
Wash 3x with 0.05% Tween 20 / PBS
12 standard dilutions of hirudin, starting with
500 ng / l 0, 1% BSA / PBS in 2-steps, of which each
0.1 ml / well; then addition of 0.1 ml / well poly-anti-hirudin (1 ug / ml 0.1% BSA / PBS) and
Overnight incubation / 4 ° C
Washing as above,
0.2 ml / well biotinylated anti-rabbit IgG anti-body (1: 5000) for 2 h at room temperature, - washing as above,
0.2 ml / well streptavidin-peroxidase complex (1: 10,000);
30 min at room temperature
Washing as above,
0.1 ml / well peroxidase substrate - stop reaction with 0.1 ml / well 2M H 2 Sθ
Measuring the absorbance at 450 nm
The obtained OD values were plotted against the logarithm of
Standard dilutions were applied and in the range of about 60 to 2 ng / ml, a linear dependence. Samples of unknown concentration of hirudin is set parallel to the calibration sample in different dilutions in a test. In the measured samples, hirudin was detected, which corresponds to an activity of maximum 5 ATU / ml.
b) thrombin time (= TZ)
In the analysis of the new protein, the measurement of TZ gave a antithrombin activity 200 ATU / ml. This value was a factor of> 40 over by ELISA for hirudin er¬ mediated value. This proves that the new protein is hirudin.
An attempt was made to focus hirudin using Centricon membranes at p and the seventh Hirudin was not retained by the Centricon 10-Memb. The new protein is retained under these Condition E of the membrane (see FIG. 4a).
Priority Applications (2)
|Application Number||Priority Date||Filing Date||Title|
|DE19904031731 DE4031731A1 (en)||1990-10-06||1990-10-06||Protein from hirudo medicinalis|
|Publication Number||Publication Date|
|EP0551320A1 true true EP0551320A1 (en)||1993-07-21|
Family Applications (1)
|Application Number||Title||Priority Date||Filing Date|
|EP19910916983 Withdrawn EP0551320A1 (en)||1990-10-06||1991-09-27||Protein produced from hirudo medicinalis|
Country Status (5)
|EP (1)||EP0551320A1 (en)|
|JP (1)||JPH06501255A (en)|
|CA (1)||CA2093432A1 (en)|
|DE (1)||DE4031731A1 (en)|
|WO (1)||WO1992006118A1 (en)|
Family Cites Families (1)
|Publication number||Priority date||Publication date||Assignee||Title|
|EP0209061B1 (en) *||1985-07-17||1994-01-12||Hoechst Aktiengesellschaft||Peptides having an anticoagulant activity, process for their preparation, obtention, their use and agents containing them|
Non-Patent Citations (1)
|See references of WO9206118A1 *|
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